(1) Field of the Invention
This invention relates generally to methods and compositions for the extraction of nucleic acids from biological samples, and, more particularly, to methods and compositions for rapid extraction of nucleic acids from tissue samples using an alkaline solution containing proteinase K. The extraction solution is suitable for further processing of the extracted nucleic acid using PCR.
(2) Description of the Related Art
With the advent of modern molecular biology, the ability to study nucleic acids in biological samples has allowed many significant advances in biological and biochemical research. One method that has provided such advances has been the polymerase chain reaction (PCR) which allows the rapid amplification of target nucleic acid from as little starting material as a single molecule (for review see Baumforth et al, J. Clin. Pathol. Mol. Pathol. 52:1–10, 1999; Rapley et al, Medical Laboratory Sciences 49:119–128, 1992).
The application of PCR and other methods in molecular biology require the extraction of nucleic acid from biological samples and a number of approaches have been devised for performing such extraction. Various approaches have included treatment with a surface active agent such as sodium dodecyl sulfate and proteinase K to lyse cells and release the nucleic acid along with extraction using phenol and/or chloroform (see, for example, Sambrook et al., Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
In recent years, a number of approaches have been developed for rapid extraction of nucleic acids from biological samples. The methods not only provide an ease and convenience of tissue processing, they allow the processing of a high volume of samples (see for example, Steiner et al, Nucleic Acids Research 23:2569–2570, 1995). Nevertheless, efficient extraction of biological samples has not always been achieved.
Both animal and plant tissues have been studied and approaches for nucleic acid extraction have been developed for both. For mammalian tissue extraction, some studies have reported on the digestion of the tissue by incubation with proteinase K for hours at elevated temperatures (see for example, Goldenberger et al., PCR Methods and Applications 4: 368–370, 1995; Zimmermann et al., Comparative Medicine 50:314–316, 2000) Such methods can form the basis for kits, which are commercially available (see for example GenElute™ Mammalian Genomic DNA Miniprep kit available from Sigma-Aldrich, St. Louis Mo.).
Other studies have reported shorter incubation times, which are more applicable for use in high throughput assays. For example, U.S. Pat. No. 6,469,159 discloses an extraction method using a buffer, a non-ionic surfactant and heating at alkaline pH. Heating was to 70° C. to 100° C. for 5 minutes to 3 hours. This reference, however, did not disclose extraction at temperatures less than 70° C., which could have been more conveniently performed or the use of an extraction solution, which did not contain a surfactant.
Drews et al. reported on a 15 minute procedure for extraction of mouse tail sections at 55° C. The procedure used a Tris-HCl buffer at pH 8.0 and containing the ionic surfactant, sodium dodecyl sulfate (SDS), and Proteinase K (Drews et al., BioTechniques 17:866–867, 1994). Similarly, Chen et al. reported on a 30 min procedure for extracting mouse ear-punch tissues at 55° C. using a Tris-HCl buffer at pH 8.0, SDS and Proteinase K (Chen et al., BioTechniques 8:32–33, 1990). Although SDS is known to inhibit Taq polymerase in PCR reactions (e.g. see Gelfand, in PCR Technology, H. A. Erlich, Ed., Stockton Press, N.Y., 1989 pp. 17–22), these groups provided no suggestion that extraction could be carried out without surfactant. In addition, there was no suggestion in these references that extraction of the mouse tissues might have been carried out at room temperature, which would have been more convenient.
Ren et al, however, reported on the extraction of mouse ear-punch tissue using a detergent-free, proteinase K solution in sterile water at room temperature for 30 min (Ren et al., Contemp. Top. Lab. Anim. Sci. (US) 40:27–30, 2001). This group, however, did not use a Tris-HCl buffer and particularly noted the absence of strong bases and acids in their extraction solution.
Methods for rapid extraction of plant tissues have also been reported. Thomson et al. reported on the extraction of DNA from ground leaf, seeds and embryos using a Tris-HCl buffer at pH 9.5 and incubation at 95° for 5–60 min and at 65° for 10–60 min (Thomson et al, BioTechniques 19:394–400, 2002). McCarthy et al. reported on the extraction of DNA from ground transgenic wheat seeds at room temperature using an extraction buffer containing urea, SDS and EDTA. A 1:1 phenol:chloroform mixture was then added to the extraction buffer (BioTechniques 32:560–564, 2002). Steiner et al. reported on the extraction of lyophilized and ground leaf tissue at 90° for 20 min using an extraction buffer containing Tris-HCl at pH 8, sodium lauryl sarkosyl and polyvinylpolypyrrolidone. None of these groups reported on the use of a protease enzyme such as Proteinase K in the extraction buffer.
Guidet reported incorporating Proteinase K into the extraction buffer to extract DNA from lyophilized and crushed leaf samples (Guidet, Nucleic Acids Res. 21:4153–4154, 1994). The buffer contained Tris-HCl at pH 8, EDTA, sodium lauryl sarkosyl and Proteinase K and the extraction was at 50° C. for 1 hour. The Proteinase K, however, may not have significantly contributed to the extraction since the EDTA, which is known to be a Ca2+ chelator, was present at a concentration of 450 mM. This is because Proteinase K is Ca2+-dependent in that the enzyme is unstable at the high temperatures used by Guidet as well as exhibiting a decrease in enzyme activity in the absence of Ca2+ (Bajorath et al., Nature 337:481–484, 1989; Muller et al, J. Biol. Chem. 269:23108–23111; Kolvenbach et al., Int. J. Pept. Protein Res 36:387–391, 1990). Moreover, the detergent, sodium lauryl sarkosyl is present which may affect subsequent PCR amplification (Gelfand, supra, 1989).
Thus, in view of the deficiencies of earlier methods and compositions, there remains a continuing need for improved methods and compositions for extracting nucleic acids from plant and animal samples.